1. Technical Field of the Invention
The present invention relates to the synthesis of peptide analogs. More particularly, the invention relates to a novel polymeric disc, wafer or other similarly shaped resin and a method for its use in solid phase peptide synthesis ("SPPS").
The present invention permits the rapid production of peptide analogs, i.e., numerous peptides differing from one another by only a single amino acid or a small number of amino acids. The synthesis of analogs according to the present invention can take place at a rapid rate while assuring that the reagents necessary to synthesize the analogs undergo quantitatively complete reactions so as to minimize undesirable side-reaction products which could result in the production of "deletion peptides" or "deletion sequences."
Within recent years, new hormones, releasing factors, inhibitors, growth factors, toxins, ion carriers and antibiotics have been discovered. This and related activity has created an increased need for the chemical synthesis of peptides and small proteins. Synthetic peptide analogs are essential for structure-function studies designed to investigate the mechanism of action and to produce inhibitors or superagonists of improved selectivity and duration of action. The synthesis of immunogenic peptides has great potential for the development of vaccines and can play an important role in the detection and isolation of new gene products. The present invention greatly simplifies and increases the efficiency of the task of preparing synthesis peptides.
2. Description of the Prior Art
Solid phase peptide synthesis was introduced by Dr. R. Bruce Merrifield in 1963 when Dr. Merrifield attached a growing peptide chain to a solid support. Merrifield, R. B. (1963) J. Am. Chem. Soc. 85, 2149-2154. The procedures enunciated by Dr. Merrifield for SPPS were as follows: An amino acid corresponding to the C-terminal of the target peptide is covalently attached to an insoluble polymeric support (the "resin"). The next amino acid, with a protected .alpha.-amino acid, is activated and reacted with the resin-bound amino acid to yield an amino protected dipeptide on the resin. Excess reactants and co-products are removed by filtration and washing. The amino-protecting group is removed and chain extension is continued with the third and subsequent protected amino acids. After the target protected peptide chain has been built up in this stepwise fashion, all side chain groups are removed and the anchoring bond between the peptide and the resin is cleaved by suitable chemical means thereby releasing the crude peptide product into solution. The desired peptide then undergoes an extensive purification procedure and is then characterized. Kent, S. & Clark-Lewis, I., "Modern Methods for the Chemical Synthesis of Biologically Active Peptides," Division of Biology 147-75, California Institute of Technology, Pasadena, Calif. 91125 U.S.A.; Houghten, R. A., Chang, W. C. & Li, C. H. (1980), Int. J. Pept. Protein Res., 16, 311-320; Houghten, R. A., Ostresh, J. M. & Klipstein, F. A. (1984), Eur. J Biochem., 145, 157-162; Stewart, J. M. & Young, J. D., Solid Phase Peptide Synthesis, Pierce Chemical Company (2d ed. 1984). See, also, Geysen, H. M., Meloen, R. H. & Barteling, S. J. (1984) Proc. Natl. Acad. Sci. USA, 81, 3998-4002; Matthes, H. W. D., Zenke, W. M., Grundstrom, T., Staub, A., Wintzerith, M. & Chambon P., (1984) The EMBO Journal, 3, 801-805.
The resin employed in standard SPPS is known as the "Merrifield resin" and is a polystyrene bead of 100-200 microns in size. The resin typically contains 0.5-2.0% divinylbenzene cross-linkage and contains 0.2 to 0.8 mmole of p-chloromethyl groups per gram resin. The number of p-chloromethyl groups determines the number of individual chains per gram and their ultimate size. The size of the bead allows for a rapid penetration of reagents in SPPS. The percentage of cross-linkage determines the extent to which the resin shrinks and swells during solvent changes. A large shrink-and-swell effect is preferred.
Dr. Merrifield had adopted known techniques of peptide chemistry, which were being used by others in solution phase peptide synthesis, for solid phase peptide synthesis. In doing so, Dr. Merrifield eliminated the intensive purification procedures required between each chemical step; the solid-phase procedure only required filtration and rinsing of the solid support with fresh solvent. Solid phase synthesis permitted chemists to add 5-6 amino acids per day rather than one or two amino acids per week.
While the solid phase technique had revolutionized biomedical research in industry and academia, this procedure has remained essentially unchanged since its inception in the early 1960's. With the explosive pace at which biotechnical research has been advancing in the industralized nations of the world, substantially more peptides, particularly analogs, of greater complexity are needed in industry and research than ever before.
The ever increasing demand for analog peptides has been approached in several ways, but no approach, thus far, has proven completely satisfactory. One highly expensive and labor intensive method has been to use a series of reaction vessels, e.g., Stewart, J. M. & Young, J. D., Solid Phase Peptide Synthesis, Pierce Chemical Company, pp. 125-130 (2d ed. 1984), rather than use of a single reaction vessel.
Thereafter, the "pin" method was developed which resulted in the synthesis of peptides on the surface of a dowel rod. The concept was to employ many rods along a plate with each rod entering a different reaction well. The drawbacks inherent in the pin method are multi-fold. First, the formed peptide remains on the dowel during biological testing; there is no guarantee that the conformation of the bound peptide duplicate the conformation in solution. Secondly, and more importantly, each analog in actuality represented a separate synthesis. Accordingly, if one dowel were to show a superior biological response, there would be no means of determining whether the reactions involved in the synthesis of a particular analog was superior or whether an analog synthesized was biologically superior.
Subsequent to the pin method, the "tea bag" method was developed where a resin was placed in individual packets similar in design to ordinary tea bags. See, Houghten, R. A., (1985) Proc. Natl. Acad. Sci. USA 82, 5131-5135. The concept of the tea bag method was that many tea bags could be placed into the same reaction vessel so that many peptides could be synthesized together. When the point of difference or deviation was reached in the formation of particular peptides, i.e., the point where an analog would differ from a similar peptide by a single or small number of amino acids, each tea bag would be separated by hand and reacted separately for the differing amino acids. Following the necessary separate reactions, the tea bags would all be returned to the same reaction vessel for the continued formation of those portions of the analogs which would be common to several peptides, thereby minimizing experimental error. Theoretically and initially, the tea bag method appeared to be ideal. In practice, however, the tea bag mesh would necessarily be prohibitively small. The flow of reagents to the resin would be inhibited. Consequently, many reactions would fail to go to completion thereby resulting in the synthesis of peptides having deletion sequences. The resulting truncated peptides could not, without possibly great difficulty, be separated from analog peptides having the proper sequence.